An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein

Abstract

Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with ∼10% protein and up to 40% lignin. These wall polymers interact covalently and noncovalently to form the functional cell wall. Characterized cross-links in the wall include covalent linkages between wall glycoprotein extensins between rhamnogalacturonan II monomer domains and between polysaccharides and lignin phenolic residues. Here, we show that two isoforms of a purified Arabidopsis thaliana arabinogalactan protein (AGP) encoded by hydroxyproline-rich glycoprotein family protein gene At3g45230 are covalently attached to wall matrix hemicellulosic and pectic polysaccharides, with rhamnogalacturonan I (RG I)/homogalacturonan linked to the rhamnosyl residue in the arabinogalactan (AG) of the AGP and with arabinoxylan attached to either a rhamnosyl residue in the RG I domain or directly to an arabinosyl residue in the AG glycan domain. The existence of this wall structure, named ARABINOXYLAN PECTIN ARABINOGALACTAN PROTEIN1 (APAP1), is contrary to prevailing cell wall models that depict separate protein, pectin, and hemicellulose polysaccharide networks. The modified sugar composition and increased extractability of pectin and xylan immunoreactive epitopes in apap1 mutant aerial biomass support a role for the APAP1 proteoglycan in plant wall architecture and function.

Figure 1.

Reverse-Phase HPLC Chromatography of APAP1 and Separation of Hyp-O-Glycosides from APAP1 by Size Exclusion Chromatography. (A) Reverse-phase PRP-1 chromatography profile of AGP-enriched material from Arabidopsis suspension culture medium (modified from Xu et al., 2008). AGPs from the medium of 10-d-old Arabidopsis suspension-cultured cells were purified by sequential DEAE anion exchange and Superose-12 SEC and separated into seven 220 nm-absorbing peaks by reverse-phase PRP-1 chromatography (Xu et al., 2008). The yield of peak 3 (5 mg/5 liters media) was <1% (w/w) of the total AGPs recovered. (B) The material in peak 3 from (A) was treated with Yariv reagent to yield a Yariv precipitant and a Yariv-soluble fraction. The Yariv-soluble fraction was separated by PRP-1 reverse-phase chromatography into two subfractions, major peak YS1 (1 mg/5 liters media) and shoulder peak YS2 (0.6 to 0.7 mg/5 liters media). (C) YS1 was hydrolyzed with 0.44 n NaOH at 105°C for 18 h, each hydrolysate was neutralized with 1 n HCl on ice, and freeze-dried residues were dissolved and separated over an analytical Superdex-75 column as described (see Methods). An aliquot (10% volume) of each collected fraction was assayed for Hyp and pentose. Red squares represent absorbance at 560 nm to detect Hyp derivatives; blue diamonds represent absorbance at 665 nm to detect pentose derivatives. The asterisks show the fractions used for more extensive sugar analyses (red asterisks, fractions 22 and 50) and NMR analyses (blue asterisks, fractions 26 and 27). (D) YS2 hydrolyzed and analyzed as described in (C).

Figure 2.

Protein Sequences Deduced from YS1 and YS2 Exactly Match a Portion of the Arabidopsis Gene At3g45230. (A) The gene encodes a Ser/Pro-rich AGP, At-AGP57C. N-terminal sequencing of YS1 yielded a 20–amino acid sequence that matched the underlined sequence of the mature Ser/Pro-rich protein. O stands for Hyp, as confirmed by amino acid sequencing. The double underlined sequence matched the N-terminal sequence of anhydrous HF deglycosylated YS2. (B) Domain structure diagram of At-AGP57C. Red indicates two predicted transmembrane domains (TM) of APAP1 calculated by the transmembrane prediction programs TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) and TMpred (http://www.ch.embnet.org/software/TMPRED_form.html). The TM1 regions include the predicted N-terminal TM from amino acids 5 to 22 and the predicted C-terminal TM, TM2, from amino acids 135 to 154. Green indicates non-Pro/Hyp-rich regions, both predicted to have the opposite topology of the Pro/Hyp-rich domain (marked in blue). The N-terminal TM was predicted to be a signal peptide by SignalP (http://www.cbs.dtu.dk/services/SignalP). The C-terminal TM indicated a possible signal sequence for GPI anchor modification (http://mendel.imp.ac.at/gpi/plant_server.html).

Figure 3.

NMR Spectra of YS1-HP2627 Collected at 25°C on a Varian VNMRS 800 Instrument. (A) HSQC spectrum. The inset is the enlarged anomeric region. Signals A and B were identified as the anomeric C/H of α-l-Araf residues; C, as the anomeric C/H of the 1,3-galactan backbone Gal (Gal_bb); D, as the anomeric C/H of the AG side chain Gal (Gal_sc); E, as the anomeric C/H of β-d-Xylp residues; F, as the anomeric C/H of β-d-GlcAp; G, as the C/H-6 of α-l-Rhap residues (the signal of G is folded in this HSQC spectrum due to the small ¹³C spectral width used; as such, 80 ppm should be subtracted from the respective ¹³C chemical shifts); H, as the C/H-4 of 4-Xylp. (B) COSY spectrum (partial) collected at 25°C. The correlation between Hyp H-3ax and Hyp H-3ex is labeled. (C) TOCSY spectrum identified the protons of Xyl residues, GlcA, and the Gal residue attached to Hyp. The labels X1 to X5 represent H-1 to H-5 of 4-Xyl; GA3 and GA5 represent H-3 and H-5 of GlcA; and G₁2 represents H-2 of Gal attached to Hyp. A similar pattern was also observed in TOCSY recorded on YS2-HP2627. (D) NOESY spectrum (partial) (mixing time 100 ms). Cross-peak A shows the NOE between Xyl H-1 to Ara H-5, which established the β-d-Xylp-1→5-α-l-Araf linkage; cross-peak B shows NOEs between Gal_bb H1 and Gal_bb H3, suggesting the Gal_bb-1→3-Gal_bb linkage; cross-peak C (NOE between Gal_bb H1 and Gal_bb H6) supports the Gal_bb-1→6-Gal_bb linkage; NOEs in D suggest the Gal_sc-1→6-Gal_bb linkage. (E) HMQC spectrum (partial) collected at 30°C. Signal A represents the anomeric ¹H/¹³C signals of 2-α-l-Rha, B the anomeric ¹H/¹³C signals of α-d-GalA, and C to F the anomeric ¹H/¹³C signals of α-l-Araf. Hyp-glycoside YS1-HP2627 produced by SEC chromatography of base hydrolyzed YS1 as depicted in Figure 1C.

Figure 4.

Proposed Structural Model for APAP1. RG-I is attached to type II AG through an α-d-GalA-1→2-α-l-Rha-1→4-β-d-GlcA-1→6-Gal structural unit (box 4). HG, with at least four to five GalA residues is embedded within RG-I (box 6). The length of flanking RG-I and HG domains is unknown. Arabinoxylan1 is attached to type II AG through a β-d-Xylp-1→5-α-l-Araf- linkage (box 9). However, only one out of three possible types of Ara residues (based on the common sizes of the arabinosyl side chains: Ara₁, Ara₂, and Ara₃) for this attachment is shown in this model. Furthermore, some of the Xyl residues in arabinoxylan1 are arabinosylated at position 2. Arabinoxylan2 is attached to RG-I through a β-d-Xylp-1→4-α-l-Rha linkage (box 10). The AGP protein core in APAP1 is AGP57C encoded by At3g45230. Bolded blue numbers (1 to 10) represent the identified structural units as listed in Table 2. Based on the amount of Hyp and monosaccharides in YS1 and YS2, a given Hyp residue in At-AGP57C in YS1 is attached with, on average, ∼24 Gal, 45 Ara, 28 Xyl, seven Rha, and 13 GalA/GlcA, while in YS2, there are 11 Gal, 65 Ara, 65 Xyl, nine Rha, and 13 GalA/GlcA residues. Due to the heterogeneity of glycosylation, this proposed model reflects representative, and not exact, numbers and length of the AG-pectin-(arabino)xylan chains in APAP1.

Figure 5.

Glycome Profiling of Sequential Cell Wall Extracts from 8-Week-Old Arabidopsis apap1-3 and apap1-4 Mutant and Wild-Type Plants. (A) Glycome profiling of the wild type. Panels show analyses of representative examples from multiple biological replicates (see Supplemental Figure 7 online for full set of results). Sequential cell wall extracts were prepared from the aerial portion (above the rosette leaves) of 8-week-old Arabidopsis wild-type (Columbia-0) (A), apap1-3 mutant (B), and apap1-4 mutant (C) plants. Labels at the bottom show reagents used for the different extraction steps. The amounts of material extracted in each extraction step are indicated in the bar graphs above the heat maps. Extracts were ELISA screened using 155 plant cell wall glycan-directed monoclonal antibodies (see Supplemental Table 4 online; Pattathil et al., 2010). Data are represented as heat maps. The panel on the right of the heat maps shows the antibodies used, color-coded as groups based on the principal cell wall glycans recognized by each antibody group (see Supplemental Table 4 online; Pattathil et al., 2010). Major changes in binding of specific antibodies to different mutant versus wild-type wall extracts are outlined in green (xylan groups 3 to 7) and red (HG backbone-1 and RG-1 backbone). The strength of the ELISA signal is indicated by a yellow-black scale, with bright yellow depicting strongest binding and black indicating no binding. (B) Glycome profiling of the apap1-3 mutant as described in (A). (C) Glycome profiling of the apap1-4 mutant as described in (A).